Livestock animals with improved growth performance

ABSTRACT

Livestock animals and progeny thereof comprising at least one edited chromosomal sequence that alters expression or activity of a somatostatin receptor (SSTR) protein are provided. Livestock animal cells that contain such edited chromosomal sequences are also provided. The livestock animals have improved growth performance and weight gain. Methods for producing livestock animals with increased growth performance are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to provisional application U.S. Ser.No. 62/957,861, filed Jan. 7, 2020, which is incorporated herein byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 4, 2021, isnamed 2021-01-04_ROSS_P13042US01_SEQLISTING_ST25.txt and is 16,515 bytesin size.

TECHNICAL FIELD

The present invention relates to genetically edited livestock animalsand the modification of somatostatin receptor genes to provide improvedgrowth performance.

BACKGROUND

Somatostatin (SST) plays a key role in the inhibition of growth hormone(GH), by inhibiting growth hormone releasing hormone (GHRH) in the brainvia negative feedback mechanisms. Although many attempts have been madein the past to skew the balance of SST and GHRH in favor of GHRH both byinhibiting SST and overexpressing GH, the ideas were eventually scrappedfor various reasons. Even a tiny increase in GH in commercial swineproduction would result in millions of dollars and thousands of poundsof inputs saved, and a significant reduction in the industry's carbonfootprint.

For the swine industry both in the US and abroad, raising pigs that puton more weight in less time has a huge impact on both monetary and feedinputs, as well as lowering the overall carbon footprint of theindustry. On the world stage, we are rapidly approaching a time where wewill have to produce more animal protein in less time with fewer inputs.Furthermore, demand for pork as a meat source is growing rapidly aroundthe world.

As can be seen, there is a need in the art for pigs and other livestockanimals with improved growth performance.

SUMMARY

The present invention provides livestock animals and methods forimproving growth performance by creating animals that have modifiedsomatostatin receptor (SSTR) expression or activity. The animals haveinactivated or otherwise modified SSTR expression or activity andimproved growth performance. The livestock animals can be created usingany of a number of protocols such as knock-out technology orgene-editing. Thus, an embodiment of the invention is a geneticallyedited or modified livestock animal or animal cell comprising a genomewith inactivation of a SSTR gene. In some embodiments, the modifiedsomatostatin receptor is SSTR2.

Yet another embodiment of the invention is a process of making alivestock animal comprising a livestock animal cell or livestock embryo,an agent that specifically binds to a DNA target site of the cell andcauses a double-stranded DNA break or otherwise inactivates a SSTR genetherein using gene editing methods such as the Clustered RegularlyInterspaced Short Palindromic Repeats (CRISPR)/Cas system, TranscriptionActivator-Like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFN),or recombinase fusion proteins.

Further embodiments will become evident from the detailed description ofthe invention which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the sequence alignment for 74-1. Sequence alignment fromtop to bottom: Wild type SSTR2, guide RNA target site, 10 sequencesobtained after sequencing a TA cloning vector containing the PCR productof the region of interest from 74-1 DNA. Both deletions can be seen atthe cut site between the 17th and 18th base pairs of the target site.

FIG. 2 shows the sequence alignment for 74-2. Sequence alignment fromtop to bottom: Wild type SSTR2, guide RNA target site, 10 sequencesobtained after sequencing a TA cloning vector containing the PCR productof the region of interest from 74-1 DNA. Both deletions can be seen atthe cut site between the 17th and 18th base pairs of the target site.

FIG. 3A, FIG. 3B, and FIG. 3C show SSTR2 protein sequences. FIG. 3Ashows the wild type protein sequence of SSTR2 (SEQ ID NO: 12). FIG. 3Bshows the predicted protein sequence of SSTR2 resulting from a 1 basepair deletion in exon 2 (SEQ ID NO: 13). The sequence is altered eightamino acids after the start codon and results in a premature stop codon.FIG. 3C shows the predicted protein sequence of SSTR2 resulting from a 3base pair, in frame deletion in exon 2 (SEQ ID NO: 14). The sequencelacks a single leucine eight amino acids after the start codon. FIG. 4shows daily body weight. Daily body weights recorded in the morning for74-1 and 74-2 from birth to day 19. By comparison, approximately 5.5 kgis the average body weight of a piglet at ˜18-20 days of age.

FIG. 5 shows piglets 74-1 (right) and 74-2 (left) at 21 days of age.

FIG. 6 shows weekly weight data comparing heterozygous males carryingthe 1 bp deletion (n=6) and heterozygous males carrying the 3 bpdeletion (n=12). No differences were observed between groups at birth,but differences were observed at all other time points (*, P<0.05; #,P<0.10).

FIG. 7 shows weekly weight data comparing heterozygous females carryingthe 1 bp deletion (n=10) and heterozygous females carrying the 3 bpdeletion (n=8). No differences were observed between groups at any ofthe time points.

DETAILED DESCRIPTION

The present invention now will be described more fully with reference tothe accompanying examples. The invention may be embodied in manydifferent forms and should not be construed as limited to theembodiments set forth in this application; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains, havingthe benefit of the teachings presented in the descriptions and thedrawings herein. As a result, it is to be understood that the inventionis not to be limited to the specific embodiments disclosed and thatmodifications and other embodiments are intended to be included withinthe scope of the appended claims. Although specific terms are used inthe specification, they are used in a generic and descriptive sense onlyand not for purposes of limitation.

Units, prefixes, and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written from left to rightin 5′ to 3′ orientation; amino acid sequences are written from left toright in amino to carboxy orientation, respectively. Numeric rangesrecited within the specification are inclusive of the numbers definingthe range and include each integer within the defined range. Amino acidsmay be referred to herein by either their commonly known three lettersymbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes. The termsdefined below are more fully defined by reference to the specificationas a whole.

The singular terms “a”, “an”, and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicate otherwise.The word “or” means any one member of a particular list and alsoincludes any combination of members of that list.

By “amplified” is meant the construction of multiple copies of a nucleicacid sequence or multiple copies complementary to the nucleic acidsequence using at least one of the nucleic acid sequences as a template.Amplification systems include the polymerase chain reaction (PCR)system, ligase chain reaction (LCR) system, nucleic acid sequence basedamplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicasesystems, transcription-based amplification system (TAS), and stranddisplacement amplification (SDA). See, e. g., Diagnostic MolecularMicrobiology: Principles and Applications, D. H. Persing et al., Ed.,American Society for Microbiology, Washington, D. C. (1993). The productof amplification is termed an amplicon.

The term “Cas” refers to a “CRISPR associated” protein. Non-limitingexamples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5,Cash, Cas7, Cas8, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5,Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1,Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1,Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof.

“Cas9” (formerly referred to as Cas5, Csn1, or Csx12) herein refers to aCas endonuclease of a type II CRISPR system that forms a complex with acrNucleotide and a tracrNucleotide, or with a single guidepolynucleotide, for specifically recognizing and cleaving all or part ofa DNA target sequence.

The term “conservatively modified variants” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids which encode identical or conservatively modified variants of theamino acid sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenprotein. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations” and represent onespecies of conservatively modified variation. Every nucleic acidsequence herein that encodes a polypeptide also, by reference to thegenetic code, describes every possible silent variation of the nucleicacid.

One of ordinary skill will recognize that each codon in a nucleic acid(except AUG, which is ordinarily the only codon for methionine; and UGG,which is ordinarily the only codon for tryptophan) can be modified toyield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide of the presentinvention is implicit in each described polypeptide sequence and iswithin the scope of the present invention.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Thus, any number of amino acid residues selected from the group ofintegers from 1 to 15 can be so altered. Thus, for example, 1, 2, 3, 4,5, 7, or 10 alterations can be made.

Conservatively modified variants typically provide similar biologicalactivity as the unmodified polypeptide sequence from which they arederived. For example, substrate specificity, enzyme activity, orligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%,80%, or 90% of the native protein for its native substrate. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art.

The following six groups each contain amino acids that are conservativesubstitutions for one another: [1] Alanine (A), Serine (S), Threonine(T); [2] Aspartic acid (D), Glutamic acid (E); [3] Asparagine (N),Glutamine (Q); [4] Arginine (R), Lysine (K); [5] Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and [6] Phenylalanine (F), Tyrosine(Y), Tryptophan (W). See also, Creighton (1984) Proteins W. H. Freemanand Company.

A “CRISPR system” refers collectively to transcripts and other elementsinvolved in the expression of or directing the activity ofCRISPR-associated (“Cas”) genes, including sequences encoding a Casgene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or anactive partial tracrRNA), a tracr-mate sequence (encompassing a “directrepeat” and a tracrRNA-processed partial direct repeat in the context ofan endogenous CRISPR system), a guide sequence (also referred to as a“spacer” in the context of an endogenous CRISPR system), or othersequences and transcripts from a CRISPR locus.

By “encoding” or “encoded”, with respect to a specified nucleic acid, ismeant comprising the information for translation into the specifiedprotein. A nucleic acid encoding a protein may comprise interveningsequences (e.g., introns) within translated regions of the nucleic acid,or may lack such intervening non-translated sequences (e.g., as incDNA). The information by which a protein is encoded is specified by theuse of codons. Typically, the amino acid sequence is encoded by thenucleic acid using the “universal” genetic code. When the nucleic acidis prepared or altered synthetically, advantage can be taken of knowncodon preferences of the intended host where the nucleic acid is to beexpressed.

As used herein “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire amino acidsequence of a native (nonsynthetic), endogenous, biologically activeform of the specified protein. Methods to determine whether a sequenceis full-length are well known in the art including such exemplarytechniques as northern or western blots, primer extension, S1protection, and ribonuclease protection. Comparison to known full-lengthhomologous (orthologous and/or paralogous) sequences can also be used toidentify full-length sequences of the present invention. Additionally,consensus sequences typically present at the 5′ and 3′ untranslatedregions of mRNA aid in the identification of a polynucleotide asfull-length. For example, the consensus sequence ANNNNAUGG, where theunderlined codon represents the N-terminal methionine, aids indetermining whether the polynucleotide has a complete 5′ end. Consensussequences at the 3′ end, such as polyadenylation sequences, aid indetermining whether the polynucleotide has a complete 3′ end.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous structural gene isfrom a species different from that from which the structural gene wasderived, or, if from the same species, one or both are substantiallymodified from their original form. A heterologous protein may originatefrom a foreign species or, if from the same species, is substantiallymodified from its original form by deliberate human intervention.

By “host cell” is meant a cell which contains a vector and supports thereplication and/or expression of the vector. Host cells may beprokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, amphibian, or mammalian cells.

The term “hybridization complex” includes reference to a duplex nucleicacid structure formed by two single-stranded nucleic acid sequencesselectively hybridized with each other.

The term “introduced” in the context of inserting a nucleic acid into acell is equivalent to “transfection” or “transformation” or“transduction,” and includes reference to the incorporation of a nucleicacid into a eukaryotic or prokaryotic cell where the nucleic acid may beincorporated into the genome of the cell (e. g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

The term “isolated” refers to material, such as a nucleic acid or aprotein, which is: (1) substantially or essentially free from componentsthat normally accompany or interact with it as found in its naturallyoccurring environment—the isolated material optionally comprisesmaterial not found with the material in its natural environment; or (2)if the material is in its natural environment, the material has beensynthetically altered by deliberate human intervention to a compositionand/or placed at a location in the cell (e.g., genome or subcellularorganelle) not native that material. The alteration to yield thesynthetic material can be performed on the material within, or removedfrom its natural state. For example, a naturally occurring nucleic acidbecomes an isolated nucleic acid if it is altered, or if it istranscribed from DNA which has been altered, by means of humanintervention performed within the cell from which it originates. See,e.g., Compounds and Methods for Site Directed Mutagenesis in EukaryoticCells, Kmiec, U.S. Pat. No. 5,565,350; In Vivo Homologous SequenceTargeting in Eukaryotic Cells; Zarling et al., PCT/US93/03868. Likewise,a naturally occurring nucleic acid (e.g., a promoter) becomes isolatedif it is introduced by non-naturally occurring means to a locus of thegenome not native to that nucleic acid. Nucleic acids which are“isolated” as defined herein, are also referred to as “heterologous”nucleic acids.

As used herein, “marker” includes reference to a locus on a chromosomethat serves to identify a unique position on the chromosome. A“polymorphic marker” includes reference to a marker which appears inmultiple forms (alleles) such that different forms of the marker, whenthey are present in a homologous pair, allow transmission of each of thechromosomes of that pair to be followed. A genotype may be defined byuse of one or a plurality of markers.

As used herein, “mutation” includes reference to alterations in thenucleotide sequence of a polynucleotide, for example a gene or codingDNA sequence (CDS), compared to the wild-type sequence. The termincludes, without limitation, substitutions, insertions, frameshifts,deletions, inversions, translocations, duplications, splice-donor sitemutations, point-mutations or the like.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single-ordouble-stranded form, and unless otherwise limited, encompassesconservatively modified variants and known analogues having theessential nature of natural nucleotides in that they hybridize tosingle-stranded nucleic acids in a manner similar to naturally occurringnucleotides (e. g., peptide nucleic acids).

By “nucleic acid library” is meant a collection of isolated DNA or RNAmolecules which comprise and substantially represent the entiretranscribed fraction of a genome of a specified organism. Constructionof exemplary nucleic acid libraries, such as genomic and cDNA libraries,is taught in standard molecular biology references such as Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology,Vol. 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook etal., Molecular Cloning-A Laboratory Manual, 2nd ed., Vol. 1-3 (1989);and Current Protocols in Molecular Biology, F. M. Ausubel et al., Eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc. (1994).

As used herein “operably linked” includes reference to a functionallinkage between a promoter and a second sequence, wherein the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence. Generally, operably linked meansthat the nucleic acid sequences being linked are contiguous and, wherenecessary join two protein coding regions, contiguously and in the samereading frame.

As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide, or conservatively modifiedvariants; the term may also refer to analogs thereof that have theessential nature of a natural ribonucleotide in that they hybridize,under stringent hybridization conditions, to substantially the samenucleotide sequence as naturally occurring nucleotides and/or allowtranslation into the same amino acid(s) as the naturally occurringnucleotide(s). A polynucleotide can be full-length or a subsequence of anative or heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art.

The term polynucleotide as it is employed herein embraces suchchemically, enzymatically or metabolically modified forms ofpolynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including among other things,simple and complex cells.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms also may apply to conservatively modified variants and to aminoacid polymers in which one or more amino acid residue is an artificialchemical analogue of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers. The essential natureof such analogues of naturally occurring amino acids is that, whenincorporated into a protein, the protein is specifically reactive toantibodies elicited to the same protein but consisting entirely ofnaturally occurring amino acids. The terms “polypeptide”, “peptide” and“protein” are also inclusive of modifications including, but not limitedto, glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation. It will beappreciated, as is well known and as noted above, that polypeptides arenot always entirely linear. For instance, polypeptides may be branchedas a result of ubiquitization, and they may be circular, with or withoutbranching, generally as a result of posttranslation events, includingnatural processing event and events brought about by human manipulationwhich do not occur naturally. Circular, branched and branched circularpolypeptides may be synthesized by non-translation natural process andby entirely synthetic methods, as well. Further, this inventioncontemplates the use of both the methionine-containing and themethionine-less amino terminal variants of the protein of the invention.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. Examplesof promoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues, such astestes, ovaries, or placenta. Such promoters are referred to as “tissuepreferred”. Promoters which initiate transcription only in certaintissue are referred to as “tissue specific”. A “cell type” specificpromoter primarily drives expression in certain cell types in one ormore organs, for example, germ cells in testes or ovaries. An“inducible” or “repressible” promoter is a promoter which is underenvironmental control. Examples of environmental conditions that mayaffect transcription by inducible promoters include stress, andtemperature. Tissue specific, tissue preferred, cell type specific andinducible promoters constitute the class of “non-constitutive”promoters. A “constitutive” promoter is a promoter which is active undermost environmental conditions.

As used herein “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid or that the cell is derived from a cell so modified. Thus, forexample, recombinant cells express genes that are not found in identicalform within the native (non-recombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under-expressed ornot expressed at all as a result of deliberate human intervention. Theterm “recombinant” as used herein does not encompass the alteration ofthe cell or vector by naturally occurring events (e.g., spontaneousmutation, natural transformation/transduction/transposition) such asthose occurring without deliberate human intervention.

As used herein, a “recombinant expression cassette” is a nucleic acidconstruct, generated recombinantly or synthetically, with a series ofspecified nucleic acid elements which permit transcription of aparticular nucleic acid in a host cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed, and apromoter.

The terms “residue” or “amino acid residue” or “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide, or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and, unless otherwiselimited, may encompass non-natural analogs of natural amino acids thatcan function in a similar manner as naturally occurring amino acids.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toanother nucleic acid sequence or other biologics. When utilizing ahybridization-based detection system, a nucleic acid probe is chosenthat is complementary to a reference nucleic acid sequence, and then byselection of appropriate conditions the probe and the reference sequenceselectively hybridize, or bind, to each other to form a duplex molecule.

The term “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences can be identified which are 100%complementary to the probe (homologous probing).

Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, optionally less than 500 nucleotides inlength.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e. g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Specificity istypically the function of post-hybridization washes, the criticalfactors being the ionic strength and temperature of the final washsolution. For DNA/DNA hybrids, the thermal melting point (Tm) can beapproximated from the equation of Meinkoth and Wahl, Anal. Biochem.,138: 267-284 (1984): Tm [° C.]=81.5+16.6 (log M)+0.41(% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The Tm is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. Tm is reduced by about1° C. for each 1% of mismatching; thus, Tm, hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theTm can be decreased 10° C. Generally, stringent conditions are selectedto be about 5° C. lower than the Tm for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1 to 4°C. lower than the Tm; moderately stringent conditions can utilize ahybridization and/or wash at 6 to 10° C. lower than the Tm; lowstringency conditions can utilize a hybridization and/or wash at 11 to20° C. lower than the Tm. Using the equation, hybridization and washcompositions, and desired Tm, those of ordinary skill will understandthat variations in the stringency of hybridization and/or wash solutionsare inherently described. An extensive guide to the hybridization ofnucleic acids is found in Tijssen, Laboratory Techniques in Biochemistryand Molecular Biology—Hybridization with Nucleic Acid Probes, Part I,Chapter 2 “Overview of principles of hybridization and the strategy ofnucleic acid probe assays”, Elsevier, New York (1993); and CurrentProtocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995).

As used herein, “transgenic animal, cell or tissue” includes referenceto an animal which includes within its genome a heterologouspolynucleotide. Generally, the heterologous polynucleotide is stablyintegrated within the genome such that the polynucleotide is passed onto successive generations. The heterologous polynucleotide may beintegrated into the genome alone or as part of a recombinant expressioncassette.

“Transgenic” is used herein to include any cell, cell line, tissue, ororgan, the genotype of which has been altered by the presence ofheterologous nucleic acid including those transgenics initially soaltered as well as those created by sexual crosses or asexualpropagation from the initial transgenic. The term “transgenic” as usedherein does not encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional breeding methods or by naturallyoccurring events such as random cross-fertilization, non-recombinantviral infection, non-recombinant bacterial transformation,non-recombinant transposition, or spontaneous mutation.

As used herein, “vector” includes reference to a nucleic acid used intransfection of a host cell and into which can be inserted apolynucleotide. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

The following terms are used to describe the sequence relationshipsbetween a polynucleotide/polypeptide of the present invention with areference polynucleotide/polypeptide: (a) “reference sequence”, (b)“comparison window”, (c) “sequence identity”, and (d) “percentage ofsequence identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison with a polynucleotide/polypeptide of thepresent invention. A reference sequence may be a subset or the entiretyof a specified sequence; for example, as a segment of a full-length cDNAor gene sequence, or the complete cDNA or gene sequence.

(b) As used herein, “comparison window” includes reference to acontiguous and specified segment of a polynucleotide/polypeptidesequence, wherein the polynucleotide/polypeptide sequence may becompared to a reference sequence and wherein the portion of thepolynucleotide/polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. Generally, the comparison window is atleast 20 contiguous nucleotides/amino acids residues in length, andoptionally can be 30, 40, 50, 100, or longer. Those of skill in the artunderstand that to avoid a high similarity to a reference sequence dueto inclusion of gaps in the polynucleotide/polypeptide sequence, a gappenalty is typically introduced and is subtracted from the number ofmatches.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482(1981); by the homology alignment algorithm of Needleman and Wunsch,J. Mol. Biol. 48: 443 (1970); by the search for similarity method ofPearson and Lipman, Proc. Natl. Acad. Sci. 85: 2444 (1988); and bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Cali.; GAP, BESTFIT, BLAST, FASTA, and TFASTA, and relatedprograms in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). The CLUSTAL program is well described by Higgins and Sharp, Gene73: 237-244 (1988); Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet,et al., Nucleic Acids Research 16: 10881-90 (1988); Huang, et al.,Computer Applications in the Biosciences 8: 155-65 (1992), and Pearson,et al., Methods in Molecular Biology 24: 307-331 (1994).

The BLAST family of programs that can be used for database similaritysearches includes: BLASTN for nucleotide query sequences againstnucleotide database sequences; BLASTX for nucleotide query sequencesagainst protein database sequences; BLASTP for protein query sequencesagainst protein database sequences; TBLASTN for protein query sequencesagainst nucleotide database sequences; and TBLASTX for nucleotide querysequences against nucleotide database sequences. See, Current Protocolsin Molecular Biology, Chapter 19, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995); Altschul et al., J.Mol. Biol., 215: 403-410 (1990); and, Altschul et al., Nucleic AcidsRes. 25: 3389-3402 (1997). Software for performing BLAST analyses ispublicly available, for example through the National Center forBiotechnology Information (ncbi.nlm.nih.gov/). This algorithm has beenthoroughly described in a number of publications. See, e.g., Altschul SF et al., Gapped BLAST and PSI-BLAST: a new generation of proteindatabase search programs, 25 NUCLEIC ACIDS RES. 3389 (1997); NationalCenter for Biotechnology Information, THE NCBI HANDBOOK [INTERNET],Chapter 16: The BLAST Sequence Analysis Tool (McEntyre J, Ostell J,eds., 2002), available athttp://www.ncbi.nlm.nih.gov/books/NBK21097/pdf/ch16.pdf. The BLASTPprogram for amino acid sequences has also been thoroughly described (seeHenikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90: 5873-5877 (1993)). A number of low-complexity filter programs can beemployed to reduce such low-complexity alignments. For example, the SEG(Wooten and Federhen, Comput. Chem., 17: 149-163 (1993)) and XNU(Claverie and States, Comput. Chem., 17: 191-201 (1993)) low-complexityfilters can be employed alone or in combination.

Multiple alignment of the sequences can be performed using the CLUSTALmethod of alignment (Higgins and Sharp (1989) CABIOS. 5: 151-153) withthe default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the CLUSTAL method includeKTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences includes reference to theresidues in the two sequences which are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g. charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences which differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well-known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions may be calculated according to thealgorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17(1988), for example as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif., USA).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

As used herein, “gene editing,” “gene edited” “genetically edited” and“gene editing effectors” refer to the use of naturally occurring orartificially engineered nucleases, also referred to as “molecularscissors.” The nucleases create specific double-stranded break (DSBs) atdesired locations in the genome, which in some cases harnesses thecell's endogenous mechanisms to repair the induced break by naturalprocesses of homologous recombination (HR) and/or nonhomologousend-joining (NHEJ). Gene editing effectors include Zinc Finger Nucleases(ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), theClustered Regularly Interspaced Short Palindromic Repeats/Cas(CRISPR/Cas) system, and meganuclease re-engineered as homingendonucleases.

The terms “genetic manipulation” and “genetically manipulated” includegene editing techniques, as well as and/or in addition to othertechniques and processes that alter or modify the nucleotide sequence ofa gene or gene, or modify or alter the expression of a gene or genes.

As used herein “homing DNA technology” or “homing technology” covers anymechanisms that allow a specified molecule to be targeted to a specifiedDNA sequence including Zinc Finger (ZF) proteins, TranscriptionActivator-Like Effectors (TALEs), meganucleases, and CRISPR systems(e.g., CRISPR/Cas9 systems).

The term “livestock animal” includes animals traditionally raised inlivestock farming, such as beef cattle, dairy cattle, pigs, sheep,goats, horses, mules, asses, buffalo, and camels. The term also includesbirds raised commercially for meat or eggs (i.e., chickens, turkeys,ducks, geese, guinea fowl, and squabs). This does not include rats,mice, or other rodents.

As used herein “blastocyst” means an early developmental stage of embryocomprising of inner cell mass (from which embryo proper arises) and afluid filled cavity typically surrounded by a single layer oftrophoblast cells. “Developmental Biology”, sixth edition, ed. by ScottF. Gilbert, Sinauer Associates, Inc., Publishers, Sunderland, Mass.(2000)

As used herein “conditional knock-out” or “conditional mutation” meanswhen the knock-out or mutation is achieved when certain conditions aremet. These conditions include but are not limited to the presence ofcertain inducing agents, recombinases, antibiotics, and certaintemperature or salt levels.

The term “early stage embryo” means any embryo at embryonic stagesbetween fertilized ovum and blastocyst. Typically, eight cell stage andmorula stage embryos are referred to as early stage embryos.

The phrase “genetically edited” means those animals or embryos or cellswhich have a desired genetic modification such as a knock-out, knock-in,conditional, inducible, transient or point mutation(s) of any gene orits regulatory mechanism or a transgenic with foreign or modified gene/sor regulatory sequences, or having undergone genomic modification in anyway including but not limited to recombination, chromosomal deletion,addition, translocation, rearrangement or addition, deletion ormodification of nucleic acid, protein or any other natural or syntheticmolecule or organelle, or cytoplasmic or nuclear transfer, leading toinheritable changes.

As used herein, the term “knock-in” means replacement of an endogenousgene with a transgene or with same endogenous gene with some structuralmodification/s, but retaining the transcriptional control of theendogenous gene.

“Knock-out” means disruption of the structure or regulatory mechanism ofa gene. Knock-outs may be generated through homologous recombination oftargeting vectors, replacement vectors or hit-and-run vectors or randominsertion of a gene trap vector resulting into complete, partial orconditional loss of gene function. “Oogenesis” means the process ofgeneration of mature eggs from the primordial germ cells in females.

“Wild type” means those animals, embryos, or cells derived therefrom,which have not been genetically edited and are usually inbred andoutbred strains developed from naturally occurring strains.

The term “growth performance” is known in the art as a reference to thecriteria of growth rate of an animal. The “growth rate” or “weight gain”of an animal is the rate of unit gain in live weight of the animal.Growth rate or weight gain is obtained from successive measurements oflive weight over a certain period of time. Accordingly, in the presentinvention the term “growth performance” means an improvement or increasein growth rate or weight gain over time of an animal.

A “binding protein” is a protein that is able to bind to anothermolecule. A binding protein can bind to, for example, a DNA molecule (aDNA-binding protein), an RNA molecule (an RNA-binding protein) and/or aprotein molecule (a protein-binding protein). In the case of aprotein-binding protein, it can bind to itself (to form homodimers,homotrimers, etc.) and/or it can bind to one or more molecules of adifferent protein or proteins. A binding protein can have more than onetype of binding activity. For example, zinc finger proteins haveDNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains are involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit” (also referred to as a “repeat”) is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein.

Zinc finger and TALE binding domains can be “engineered” to bind to apredetermined nucleotide sequence, for example via engineering (alteringone or more amino acids) of the recognition helix region of naturallyoccurring zinc finger or TALE proteins. Therefore, engineered DNAbinding proteins (zinc fingers or TALEs) are proteins that arenon-naturally occurring. Non-limiting examples of methods forengineering DNA-binding proteins are design and selection. A designedDNA binding protein is a protein not occurring in nature whosedesign/composition results principally from rational criteria. Rationalcriteria for design include application of substitution rules andcomputerized algorithms for processing information in a database storinginformation of existing ZFP and/or TALE designs and binding data. See,for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; seealso WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO03/016496 and U.S. Publication No. 20110301073.

A “selected” zinc finger protein or TALE is a protein not found innature whose production results primarily from an empirical process suchas phage display, interaction trap or hybrid selection. See e.g., U.S.Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,200,759; WO95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO01/60970 WO 01/88197, WO 02/099084 and U.S. Publication No. 20110301073.

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 2005/0064474, 20070218528, 2008/0131962 and2011/0201055, incorporated herein by reference in their entireties.

Somatostatin Receptor Gene Editing

Somatostatin (SST) is a highly conserved peptide found in tissuesthroughout the body of many species including fish, livestock, andhumans. Through its two forms and five receptors, SST acts as a potentinhibitor of growth hormone (GH), insulin, glucagon, and thyroidstimulating hormone. Produced and released from the hypothalamus inresponse to GH levels, SST is transported to the anterior pituitarywhere it blocks the production of GH by somatotrophs and/or the blocksthe release of GH from the cells.

Various somatostatin receptor knockout mutants in mice were previouslycharacterized and no increases in growth rate or size were reported.SSTR1 gene-ablated mice exhibited significantly reduced body weight withgrowth retardation compared to wild type controls (Wang et al. 2006. MolCell Endocrinol. 247:82-90). Heterozygous SSTR2 knockouts wereindistinguishable from their normal littermates, and the homozygotesappeared normal and healthy up to 15 months of age (Zheng et al. 1997.Mol Endocrinol 11:1709-1717). In contrast to these previous studies inmice, Applicants have unexpectedly demonstrated that a SSTR2 knockoutprovides opportunity for improved growth performance in pigs that hasnot been observed in other species.

The SST receptor family of genes is known and sequences encoding thesame are available through Genbank or other such sources. Sus ScrofaSSTR2 nucleic acid and protein sequences are disclosed as SEQ ID NOs: 1and 2.

The present disclosure provides a genetically edited animal or animalcell comprising at least one edited chromosomal sequence encoding asomatostatin receptor protein. The edited chromosomal sequence may be(1) inactivated, (2) modified, or (3) comprise an integrated or deletedsequence. An inactivated chromosomal sequence is altered such that asomatostatin receptor protein function is impaired, reduced oreliminated. Thus, a genetically edited animal comprising an inactivatedchromosomal sequence may be termed a “knock out” or a “conditional knockout.” Similarly, a genetically edited animal comprising an integratedsequence may be termed a “knock in” or a “conditional knock in.”Furthermore, a genetically edited animal comprising a modifiedchromosomal sequence may comprise a targeted point mutation(s) or othermodification such that an altered protein product is produced.

In some embodiments of the present invention, at least one somatostatinreceptor locus (e.g., a SSTR2 locus) is used as a target site for thesite-specific editing. This can include insertion of an exogenousnucleic acid (e.g., a nucleic acid comprising a nucleotide sequenceencoding a polypeptide of interest) or deletions of nucleic acids fromthe locus. In particular embodiments, insertions and/or deletionsmodified locus. For example, integration of the exogenous nucleic acidand/or deletion of part of the genomic nucleic acid may modify the locusso as to produce a disrupted (i.e., inactivated) SSTR gene.

In some embodiments, the edited SSTR locus can comprise the nucleotidesequence set forth in SEQ ID NOs: 3 or 4. In some embodiments, theedited SSTR locus may comprise a nucleotide sequence that issubstantially identical to the nucleotide sequence set forth in SEQ IDNOs: 3 or 4. For example, in some embodiments, a SSTR locus is a SSTRhomologue (e.g., an ortholog or a paralog) that comprises a nucleotidesequence that is at least 85% identical to the nucleotide sequence setforth in SEQ ID NOs: 3 or 4. A SSTR homologue may comprise a nucleotidesequence that is, for example and without limitation: at least 80%; atleast 85%; at least about 90%; at least about 91%; at least about 92%;at least about 93%; at least about 94%; at least about 95%; at leastabout 96%; at least about 97%; at least about 98%; at least about 99%;at least about 99.5%; 99.6%, 99.7%, 99.8% and/or at least about 99.9%identical to about 20 contiguous nucleotides of the nucleotide sequenceset forth in SEQ ID NOs: 3 or 4. In some embodiments, the editedchromosomal sequence comprises one or more of SEQ ID NOs: 7-11.

Targeted Integration of a Nucleic Acid at a SSTR Locus

Site-specific integration of an exogenous nucleic acid at a SSTR locusmay be accomplished by any technique known to those of skill in the art.In some embodiments, integration of an exogenous nucleic acid at a SSTRlocus comprises contacting a cell (e.g., an isolated cell or a cell in atissue or organism) with a nucleic acid molecule comprising theexogenous nucleic acid. In examples, such a nucleic acid molecule maycomprise nucleotide sequences flanking the exogenous nucleic acid thatfacilitate homologous recombination between the nucleic acid moleculeand at least one SSTR locus. In particular examples, the nucleotidesequences flanking the exogenous nucleic acid that facilitate homologousrecombination may be complementary to endogenous nucleotides of the SSTRlocus. In particular examples, the nucleotide sequences flanking theexogenous nucleic acid that facilitate homologous recombination may becomplementary to previously integrated exogenous nucleotides. In someembodiments, a plurality of exogenous nucleic acids may be integrated atone SSTR locus, such as in gene stacking.

Integration of a nucleic acid at a SSTR locus may be facilitated (e.g.,catalyzed) in some embodiments by endogenous cellular machinery of ahost cell, such as, for example and without limitation, endogenous DNAand endogenous recombinase enzymes. In some embodiments, integration ofa nucleic acid at a SSTR locus may be facilitated by one or more factors(e.g., polypeptides) that are provided to a host cell. For example,nuclease(s), recombinase(s), and/or ligase polypeptides may be provided(either independently or as part of a chimeric polypeptide) bycontacting the polypeptides with the host cell, or by expressing thepolypeptides within the host cell. Accordingly, in some examples, anucleic acid comprising a nucleotide sequence encoding at least onenuclease, recombinase, and/or ligase polypeptide may be introduced intothe host cell, either concurrently or sequentially with a nucleic acidto be integrated site-specifically at a SSTR locus, wherein the at leastone nuclease, recombinase, and/or ligase polypeptide is expressed fromthe nucleotide sequence in the host cell.

DNA-Binding Polypeptides

In some embodiments, site-specific integration may be accomplished byutilizing factors that are capable of recognizing and binding toparticular nucleotide sequences, for example, in the genome of a hostorganism. For instance, many proteins comprise polypeptide domains thatare capable of recognizing and binding to DNA in a site-specific manner.A DNA sequence that is recognized by a DNA-binding polypeptide may bereferred to as a “target” sequence. Polypeptide domains that are capableof recognizing and binding to DNA in a site-specific manner generallyfold correctly and function independently to bind DNA in a site-specificmanner, even when expressed in a polypeptide other than the protein fromwhich the domain was originally isolated. Similarly, target sequencesfor recognition and binding by DNA-binding polypeptides are generallyable to be recognized and bound by such polypeptides, even when presentin large DNA structures (e.g., a chromosome), particularly when the sitewhere the target sequence is located is one known to be accessible tosoluble cellular proteins (e.g., a gene).

While DNA-binding polypeptides identified from proteins that exist innature typically bind to a discrete nucleotide sequence or motif (e.g.,a consensus recognition sequence), methods exist and are known in theart for modifying many such DNA-binding polypeptides to recognize adifferent nucleotide sequence or motif. DNA-binding polypeptidesinclude, for example and without limitation: zinc finger DNA-bindingdomains; leucine zippers; UPA DNA-binding domains; GAL4; TAL; LexA; aTet repressor; LacR; and a steroid hormone receptor.

In some examples, a DNA-binding polypeptide is a zinc finger. Individualzinc finger motifs can be designed to target and bind specifically toany of a large range of DNA sites. Canonical Cys₂His₂ (as well asnon-canonical Cys₃His) zinc finger polypeptides bind DNA by inserting anα-helix into the major groove of the target DNA double helix.Recognition of DNA by a zinc finger is modular; each finger contactsprimarily three consecutive base pairs in the target, and a few keyresidues in the polypeptide mediate recognition. By including multiplezinc finger DNA-binding domains in a targeting endonuclease, theDNA-binding specificity of the targeting endonuclease may be furtherincreased (and hence the specificity of any gene regulatory effectsconferred thereby may also be increased). See, e.g., Urnov et al. (2005)Nature 435:646-51. Thus, one or more zinc finger DNA-bindingpolypeptides may be engineered and utilized such that a targetingendonuclease introduced into a host cell interacts with a DNA sequencethat is unique within the genome of the host cell.

Preferably, the zinc finger protein is non-naturally occurring in thatit is engineered to bind to a target site of choice. See, for example,See, for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141;Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001)Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin.Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol.10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717;6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934;7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005/0064474;2007/0218528; 2005/0267061, all incorporated herein by reference intheir entireties.

An engineered zinc finger binding domain can have a novel bindingspecificity, compared to a naturally-occurring zinc finger protein.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, U.S. Pat. Nos. 6,453,242 and6,534,261, incorporated by reference herein in their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in WO 02/077227.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

Selection of target sites; ZFPs and methods for design and constructionof fusion proteins (and polynucleotides encoding same) are known tothose of skill in the art and described in detail in U.S. Pat. Nos.6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988;6,013,453; 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO98/54311; WO 00/27878; WO 01/60970 WO 01/88197; WO 02/099084; WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

In some examples, a DNA-binding polypeptide is a DNA-binding domain fromGAL4. GAL4 is a modular transactivator in Saccharomyces cerevisiae, butit also operates as a transactivator in many other organisms. See, e.g.,Sadowski et al. (1988) Nature 335:563-4. In this regulatory system, theexpression of genes encoding enzymes of the galactose metabolic pathwayin S. cerevisiae is stringently regulated by the available carbonsource. Johnston (1987) Microbiol. Rev. 51:458-76. Transcriptionalcontrol of these metabolic enzymes is mediated by the interactionbetween the positive regulatory protein, GAL4, and a 17 bp symmetricalDNA sequence to which GAL4 specifically binds (the UAS).

Native GAL4 consists of 881 amino acid residues, with a molecular weightof 99 kDa. GAL4 comprises functionally autonomous domains, the combinedactivities of which account for activity of GAL4 in vivo. Ma and Ptashne(1987) Cell 48:847-53); Brent and Ptashne (1985) Cell 43(3 Pt 2):729-36.The N-terminal 65 amino acids of GAL4 comprise the GAL4 DNA-bindingdomain. Keegan et al. (1986) Science 231:699-704; Johnston (1987) Nature328:353-5. Sequence-specific binding requires the presence of a divalentcation coordinated by 6 Cys residues present in the DNA binding domain.The coordinated cation-containing domain interacts with and recognizes aconserved CCG triplet at each end of the 17 bp UAS via direct contactswith the major groove of the DNA helix. Marmorstein et al. (1992) Nature356:408-14. The DNA-binding function of the protein positions C-terminaltranscriptional activating domains in the vicinity of the promoter, suchthat the activating domains can direct transcription.

Additional DNA-binding polypeptides that may be utilized in certainembodiments include, for example and without limitation, a bindingsequence from a AVRBS3-inducible gene; a consensus binding sequence froma AVRBS3-inducible gene or synthetic binding sequence engineeredtherefrom (e.g., UPA DNA-binding domain); TAL; LexA (see, e.g., Brent &Ptashne (1985), supra); LacR (see, e.g., Labow et al. (1990) Mol. Cell.Biol. 10:3343-56; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88(12):5072-6); a steroid hormone receptor (Ellliston et al. (1990) J.Biol. Chem. 265:11517-121); the Tet repressor (U.S. Pat. No. 6,271,341)and a mutated Tet repressor that binds to a tet operator sequence in thepresence, but not the absence, of tetracycline (Tc); the DNA-bindingdomain of NF-κB; and components of the regulatory system described inWang et al. (1994) Proc. Natl. Acad. Sci. USA 91(17):8180-4, whichutilizes a fusion of GAL4, a hormone receptor, and VP16.

In certain embodiments, the DNA-binding domain of one or more of thenucleases used in the methods and compositions described hereincomprises a naturally occurring or engineered (non-naturally occurring)TAL effector DNA binding domain. See, e.g., U.S. Patent Publication No.20110301073, incorporated by reference in its entirety herein.

In other embodiments, the nuclease comprises a CRISPR system. The CRISPR(clustered regularly interspaced short palindromic repeats) locus, whichencodes RNA components of the system, and the Cas (CRISPR-associated)locus, which encodes proteins (Jansen et al., 2002. Mol. Microbiol. 43:1565-1575; Makarova et al., 2002. Nucleic Acids Res. 30: 482-496;Makarova et al., 2006. Biol. Direct 1: 7; Haft et al., 2005. PLoSComput. Biol. 1: e60) make up the gene sequences of the CRISPR/Casnuclease system. CRISPR loci in microbial hosts contain a combination ofCas genes as well as non-coding RNA elements capable of programming thespecificity of the CRISPR-mediated nucleic acid cleavage.

The Type II CRISPR is one of the most well characterized systems andcarries out targeted DNA double-strand break in four sequential steps.First, two non-coding RNA, the pre-crRNA array and tracrRNA, aretranscribed from the CRISPR locus. Second, tracrRNA hybridizes to therepeat regions of the pre-crRNA and mediates the processing of pre-crRNAinto mature crRNAs containing individual spacer sequences. Third, themature crRNA:tracrRNA complex directs Cas9 to the target DNA viaWatson-Crick base-pairing between the spacer on the crRNA and theprotospacer on the target DNA next to the protospacer adjacent motif(PAM), an additional requirement for target recognition. Finally, Cas9mediates cleavage of target DNA to create a double-stranded break withinthe protospacer. Activity of the CRISPR/Cas system comprises of threesteps: (i) insertion of alien DNA sequences into the CRISPR array toprevent future attacks, in a process called ‘adaptation’, (ii)expression of the relevant proteins, as well as expression andprocessing of the array, followed by (iii) RNA-mediated interferencewith the foreign nucleic acid. Thus, in the bacterial cell, several Casproteins are involved with the natural function of the CRISPR/Cas systemand serve roles in functions such as insertion of the foreign DNA etc.

In certain embodiments, Cas protein may be a “functional derivative” ofa naturally occurring Cas protein. A “functional derivative” of a nativesequence polypeptide is a compound having a qualitative biologicalproperty in common with a native sequence polypeptide. “Functionalderivatives” include, but are not limited to, fragments of a nativesequence and derivatives of a native sequence polypeptide and itsfragments, provided that they have a biological activity in common witha corresponding native sequence polypeptide. A biological activitycontemplated herein is the ability of the functional derivative tohydrolyze a DNA substrate into fragments. The term “derivative”encompasses both amino acid sequence variants of polypeptide, covalentmodifications, and fusions thereof. Suitable derivatives of a Caspolypeptide or a fragment thereof include but are not limited tomutants, fusions, covalent modifications of Cas protein or a fragmentthereof. Cas protein, which includes Cas protein or a fragment thereof,as well as derivatives of Cas protein or a fragment thereof, may beobtainable from a cell or synthesized chemically or by a combination ofthese two procedures. The cell may be a cell that naturally produces Casprotein, or a cell that naturally produces Cas protein and isgenetically engineered to produce the endogenous Cas protein at a higherexpression level or to produce a Cas protein from an exogenouslyintroduced nucleic acid, which nucleic acid encodes a Cas that is sameor different from the endogenous Cas. In some case, the cell does notnaturally produce Cas protein and is genetically engineered to produce aCas protein.

In particular embodiments, a DNA-binding polypeptide specificallyrecognizes and binds to a target nucleotide sequence comprised within agenomic nucleic acid of a host organism. Any number of discreteinstances of the target nucleotide sequence may be found in the hostgenome in some examples. The target nucleotide sequence may be rarewithin the genome of the organism (e.g., fewer than about 10, about 9,about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about1 copy(ies) of the target sequence may exist in the genome). Forexample, the target nucleotide sequence may be located at a unique sitewithin the genome of the organism. Target nucleotide sequences may be,for example and without limitation, randomly dispersed throughout thegenome with respect to one another; located in different linkage groupsin the genome; located in the same linkage group; located on differentchromosomes; located on the same chromosome; located in the genome atsites that are expressed under similar conditions in the organism (e.g.,under the control of the same, or substantially functionally identical,regulatory factors); and located closely to one another in the genome(e.g., target sequences may be comprised within nucleic acids integratedas concatemers at genomic loci).

Targeting Endonucleases

In particular embodiments, a DNA-binding polypeptide that specificallyrecognizes and binds to a target nucleotide sequence may be comprisedwithin a chimeric polypeptide, so as to confer specific binding to thetarget sequence upon the chimeric polypeptide. In examples, such achimeric polypeptide may comprise, for example and without limitation,nuclease, recombinase, and/or ligase polypeptides, as these polypeptidesare described above. Chimeric polypeptides comprising a DNA-bindingpolypeptide and a nuclease, recombinase, and/or ligase polypeptide mayalso comprise other functional polypeptide motifs and/or domains, suchas for example and without limitation: a spacer sequence positionedbetween the functional polypeptides in the chimeric protein; a leaderpeptide; a peptide that targets the fusion protein to an organelle(e.g., the nucleus); polypeptides that are cleaved by a cellular enzyme;peptide tags (e.g., Myc, His, etc.); and other amino acid sequences thatdo not interfere with the function of the chimeric polypeptide.

Functional polypeptides (e.g., DNA-binding polypeptides and nucleasepolypeptides) in a chimeric polypeptide may be operatively linked. Insome embodiments, functional polypeptides of a chimeric polypeptide maybe operatively linked by their expression from a single polynucleotideencoding at least the functional polypeptides ligated to each otherin-frame, so as to create a chimeric gene encoding a chimeric protein.In alternative embodiments, the functional polypeptides of a chimericpolypeptide may be operatively linked by other means, such as bycross-linkage of independently expressed polypeptides.

In some embodiments, a DNA-binding polypeptide, or guide RNA thatspecifically recognizes and binds to a target nucleotide sequence may becomprised within a natural isolated protein (or mutant thereof), whereinthe natural isolated protein or mutant thereof also comprises a nucleasepolypeptide (and may also comprise a recombinase and/or ligasepolypeptide). Examples of such isolated proteins include TALENs,recombinases (e.g., Cre, Hin, Tre, and FLP recombinase), CRISPR systems,and meganucleases.

As used herein, the term “targeting endonuclease” refers to natural orengineered isolated proteins and mutants thereof that comprise aDNA-binding polypeptide or guide RNA and a nuclease polypeptide, as wellas to chimeric polypeptides comprising a DNA-binding polypeptide orguide RNA and a nuclease. Any targeting endonuclease comprising aDNA-binding polypeptide or guide RNA that specifically recognizes andbinds to a target nucleotide sequence comprised within a SSTR locus(e.g., either because the target sequence is comprised within the nativesequence at the locus, or because the target sequence has beenintroduced into the locus, for example, by recombination) may beutilized in certain embodiments.

Some examples of chimeric polypeptides that may be useful in particularembodiments of the invention include, without limitation, combinationsof the following polypeptides: zinc finger DNA-binding polypeptides; aFokI nuclease polypeptide; TALE domains; leucine zippers; transcriptionfactor DNA-binding motifs; and DNA recognition and/or cleavage domainsisolated from, for example and without limitation, a TALEN, arecombinase (e.g., Cre, Hin, RecA, Tre, and FLP recombinases), a CRISPRsystem, a meganuclease; and others known to those in the art. Particularexamples include a chimeric protein comprising a site-specific DNAbinding polypeptide and a nuclease polypeptide. Chimeric polypeptidesmay be engineered by methods known to those of skill in the art to alterthe recognition sequence of a DNA-binding polypeptide comprised withinthe chimeric polypeptide, so as to target the chimeric polypeptide to aparticular nucleotide sequence of interest.

In certain embodiments, the chimeric polypeptide comprises a DNA-bindingdomain (e.g., zinc finger, TAL-effector domain, etc.) and a nuclease(cleavage) domain. The cleavage domain may be heterologous to theDNA-binding domain, for example a zinc finger DNA-binding domain and acleavage domain from a nuclease or a TALEN DNA-binding domain and acleavage domain, or meganuclease DNA-binding domain and cleavage domainfrom a different nuclease. Heterologous cleavage domains can be obtainedfrom any endonuclease or exonuclease. Exemplary endonucleases from whicha cleavage domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. See, for example,2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort etal. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes whichcleave DNA are known (e.g., 51 Nuclease; mung bean nuclease; pancreaticDNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn etal. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One ormore of these enzymes (or functional fragments thereof) can be used as asource of cleavage domains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides, or nucleotidepairs, can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding, forexample, such that one or more exogenous sequences (donors/trangsenes)are integrated at or near the binding (target) sites. Certainrestriction enzymes (e.g., Type IIS) cleave DNA at sites removed fromthe recognition site and have separable binding and cleavage domains.For example, the Type IIS enzyme Fok I catalyzes double-strandedcleavage of DNA, at 9 nucleotides from its recognition site on onestrand and 13 nucleotides from its recognition site on the other. See,for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as wellas Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al.(1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc.Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem.269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise thecleavage domain (or cleavage half-domain) from at least one Type IISrestriction enzyme and one or more zinc finger binding domains, whichmay or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is Fok I. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the Fok I enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-Fok I fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a DNA binding domain and two Fok Icleavage half-domains can also be used.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in U.S. PatentPublication No. 20070134796, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the present disclosure.See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 20050064474; 20060188987 and20080131962, the disclosures of all of which are incorporated byreference in their entireties herein.

Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see e.g. U.S.Patent Publication No. 20090068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

Zinc Finger Nucleases

In some embodiments, a chimeric polypeptide is a custom-designed zincfinger nuclease (ZFN) that may be designed to deliver a targetedsite-specific double-strand DNA break into which an exogenous nucleicacid, or donor DNA, may be integrated (See US Patent publication20100257638, incorporated by reference herein). ZFNs are chimericpolypeptides containing a non-specific cleavage domain from arestriction endonuclease (for example, FokI) and a zinc fingerDNA-binding domain polypeptide. See, e.g., Huang et al. (1996) J.Protein Chem. 15:481-9; Kim et al. (1997a) Proc. Natl. Acad. Sci. USA94:3616-20; Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93:1156-60; Kimet al. (1994) Proc Natl. Acad. Sci. USA 91:883-7; Kim et al. (1997b)Proc. Natl. Acad. Sci. USA 94:12875-9; Kim et al. (1997c) Gene 203:43-9;Kim et al. (1998) Biol. Chem. 379:489-95; Nahon and Raveh (1998) NucleicAcids Res. 26:1233-9; Smith et al. (1999) Nucleic Acids Res. 27:674-81.In some embodiments, the ZFNs comprise non-canonical zinc finger DNAbinding domains (see US Patent publication 20080182332, incorporated byreference herein). The FokI restriction endonuclease must dimerize viathe nuclease domain in order to cleave DNA and introduce a double-strandbreak. Consequently, ZFNs containing a nuclease domain from such anendonuclease also require dimerization of the nuclease domain in orderto cleave target DNA. Mani et al. (2005) Biochem. Biophys. Res. Commun.334:1191-7; Smith et al. (2000) Nucleic Acids Res. 28:3361-9.Dimerization of the ZFN can be facilitated by two adjacent, oppositelyoriented DNA-binding sites. Id.

In some embodiments, a method for the site-specific integration of anexogenous nucleic acid into at least one SSTR locus of a host comprisesintroducing into a cell of the host a ZFN, wherein the ZFN recognizesand binds to a target nucleotide sequence, wherein the target nucleotidesequence is comprised within at least one SSTR locus of the host. Incertain examples, the target nucleotide sequence is not comprised withinthe genome of the host at any other position than the at least one SSTRlocus. For example, a DNA-binding polypeptide of the ZFN may beengineered to recognize and bind to a target nucleotide sequenceidentified within the at least one SSTR locus (e.g., by sequencing theSSTR locus). A method for the site-specific integration of an exogenousnucleic acid into at least one SSTR locus of a host that comprisesintroducing into a cell of the host a ZFN may also comprise introducinginto the cell an exogenous nucleic acid, wherein recombination of theexogenous nucleic acid into a nucleic acid of the host comprising the atleast one SSTR locus is facilitated by site-specific recognition andbinding of the ZFN to the target sequence (and subsequent cleavage ofthe nucleic acid comprising the SSTR locus).

Optional Exogenous Nucleic Acids For Integration at a SSTR Locus

Embodiments of the invention may include one or more nucleic acidsselected from the group consisting of: an exogenous nucleic acid forsite-specific integration in at least one SSTR locus, for example andwithout limitation, an ORF; a nucleic acid comprising a nucleotidesequence encoding a targeting endonuclease; and a vector comprising atleast one of either or both of the foregoing. Thus, particular nucleicacids for use in some embodiments include nucleotide sequences encodinga polypeptide, structural nucleotide sequences, and/or DNA-bindingpolypeptide recognition and binding sites.

Optional Exogenous Nucleic Acid Molecules For Site-Specific Integration

As noted above, insertion of an exogenous sequence (also called a “donorsequence” or “donor” or “transgene”) is provided, for example forexpression of a polypeptide, correction of a mutant gene or forincreased expression of a wild-type gene. It will be readily apparentthat the donor sequence is typically not identical to the genomicsequence where it is placed. A donor sequence can contain anon-homologous sequence flanked by two regions of homology to allow forefficient HDR at the location of interest. Additionally, donor sequencescan comprise a vector molecule containing sequences that are nothomologous to the region of interest in cellular chromatin. A donormolecule can contain several, discontinuous regions of homology tocellular chromatin. For example, for targeted insertion of sequences notnormally present in a region of interest, said sequences can be presentin a donor nucleic acid molecule and flanked by regions of homology tosequence in the region of interest.

The donor polynucleotide can be DNA or RNA, single-stranded ordouble-stranded and can be introduced into a cell in linear or circularform. See e.g., U.S. Patent Publication Nos. 20100047805, 20110281361,20110207221 and U.S. application Ser. No. 13/889,162. If introduced inlinear form, the ends of the donor sequence can be protected (e.g. fromexonucleolytic degradation) by methods known to those of skill in theart. For example, one or more dideoxynucleotide residues are added tothe 3′ terminus of a linear molecule and/or self-complementaryoligonucleotides are ligated to one or both ends. See, for example,Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls etal. (1996) Science 272:886-889. Additional methods for protectingexogenous polynucleotides from degradation include, but are not limitedto, addition of terminal amino group(s) and the use of modifiedinternucleotide linkages such as, for example, phosphorothioates,phosphoramidates, and O-methyl ribose or deoxyribose residues.

A polynucleotide can be introduced into a cell as part of a vectormolecule having additional sequences such as, for example, replicationorigins, promoters and genes encoding antibiotic resistance. Moreover,donor polynucleotides can be introduced as naked nucleic acid, asnucleic acid complexed with an agent such as a liposome or poloxamer, orcan be delivered by viruses (e.g., adenovirus, AAV, herpesvirus,retrovirus, lentivirus and integrase defective lentivirus (IDLV)).

The donor is generally integrated so that its expression is driven bythe endogenous promoter at the integration site, namely the promoterthat drives expression of the endogenous gene into which the donor isintegrated (e.g., SSTR). However, it will be apparent that the donor maycomprise a promoter and/or enhancer, for example a constitutive promoteror an inducible or tissue specific promoter.

Furthermore, although not required for expression, exogenous sequencesmay also include transcriptional or translational regulatory sequences,for example, promoters, enhancers, insulators, internal ribosome entrysites, sequences encoding 2A peptides and/or polyadenylation signals.

Exogenous nucleic acids that may be integrated in a site-specific mannerinto at least one SSTR locus, so as to modify the SSTR locus, inembodiments include, for example and without limitation, nucleic acidscomprising a nucleotide sequence encoding a polypeptide of interest;nucleic acids comprising an agronomic gene; nucleic acids comprising anucleotide sequence encoding an RNAi molecule; or nucleic acids thatdisrupt the SSTR gene.

In some embodiments, an exogenous nucleic acid is integrated at a SSTRlocus, so as to modify the SSTR locus, wherein the nucleic acidcomprises a nucleotide sequence encoding a polypeptide of interest, suchthat the nucleotide sequence is expressed in the host from the SSTRlocus. In some examples, the polypeptide of interest (e.g., a foreignprotein) is expressed from a nucleotide sequence encoding thepolypeptide of interest in commercial quantities. In such examples, thepolypeptide of interest may be extracted from the host cell, tissue, orbiomass.

Nucleic Acid Molecules Comprising a Nucleotide Sequence Encoding aTargeting Endonuclease

In some embodiments, a nucleotide sequence encoding a targetingendonuclease may be engineered by manipulation (e.g., ligation) ofnative nucleotide sequences encoding polypeptides comprised within thetargeting endonuclease. For example, the nucleotide sequence of a geneencoding a protein comprising a DNA-binding polypeptide may be inspectedto identify the nucleotide sequence of the gene that corresponds to theDNA-binding polypeptide, and that nucleotide sequence may be used as anelement of a nucleotide sequence encoding a targeting endonucleasecomprising the DNA-binding polypeptide. Alternatively, the amino acidsequence of a targeting endonuclease may be used to deduce a nucleotidesequence encoding the targeting endonuclease, for example, according tothe degeneracy of the genetic code.

In exemplary nucleic acid molecules comprising a nucleotide sequenceencoding a targeting endonuclease, the last codon of a firstpolynucleotide sequence encoding a nuclease polypeptide, and the firstcodon of a second polynucleotide sequence encoding a DNA-bindingpolypeptide, may be separated by any number of nucleotide triplets,e.g., without coding for an intron or a “STOP.” Likewise, the last codonof a nucleotide sequence encoding a first polynucleotide sequenceencoding a DNA-binding polypeptide, and the first codon of a secondpolynucleotide sequence encoding a nuclease polypeptide, may beseparated by any number of nucleotide triplets. In these and furtherembodiments, the last codon of the last (i.e., most 3′ in the nucleicacid sequence) of a first polynucleotide sequence encoding a nucleasepolypeptide, and a second polynucleotide sequence encoding a DNA-bindingpolypeptide, may be fused in phase-register with the first codon of afurther polynucleotide coding sequence directly contiguous thereto, orseparated therefrom by no more than a short peptide sequence, such asthat encoded by a synthetic nucleotide linker (e.g., a nucleotide linkerthat may have been used to achieve the fusion). Examples of such furtherpolynucleotide sequences include, for example and without limitation,tags, targeting peptides, and enzymatic cleavage sites. Likewise, thefirst codon of the most 5′ (in the nucleic acid sequence) of the firstand second polynucleotide sequences may be fused in phase-register withthe last codon of a further polynucleotide coding sequence directlycontiguous thereto, or separated therefrom by no more than a shortpeptide sequence.

A sequence separating polynucleotide sequences encoding functionalpolypeptides in a targeting endonuclease (e.g., a DNA-bindingpolypeptide and a nuclease polypeptide) may, for example, consist of anysequence, such that the amino acid sequence encoded is not likely tosignificantly alter the translation of the targeting endonuclease. Dueto the autonomous nature of known nuclease polypeptides and knownDNA-binding polypeptides, intervening sequences will not in examplesinterfere with the respective functions of these structures.

Other Knockout Methods

Various other techniques known in the art can be used to inactivategenes to make knock-out animals and/or to introduce nucleic acidconstructs into animals to produce founder animals and to make animallines, in which the knockout or nucleic acid construct is integratedinto the genome. Such techniques include, without limitation, pronuclearmicroinjection (U.S. Pat. No. 4,873,191), retrovirus mediated genetransfer into germ lines (Van der Putten et al. (1985) Proc. Natl. Acad.Sci. USA 82, 6148-1652), gene targeting into embryonic stem cells(Thompson et al. (1989) Cell 56, 313-321), electroporation of embryos(Lo (1983) Mol. Cell. Biol. 3, 1803-1814), sperm-mediated gene transfer(Lavitrano et al. (2002) Proc. Natl. Acad. Sci. USA 99, 14230-14235;Lavitrano et al. (2006) Reprod. Fert. Develop. 18, 19-23), and in vitrotransformation of somatic cells, such as cumulus or mammary cells, oradult, fetal, or embryonic stem cells, followed by nucleartransplantation (Wilmut et al. (1997) Nature 385, 810-813; and Wakayamaet al. (1998) Nature 394, 369-374). Pronuclear microinjection, spermmediated gene transfer, and somatic cell nuclear transfer areparticularly useful techniques. An animal that is genomically modifiedis an animal wherein all of its cells have the genetic modification,including its germ line cells. When methods are used that produce ananimal that is mosaic in its genetic modification, the animals may beinbred and progeny that are genomically modified may be selected.Cloning, for instance, may be used to make a mosaic animal if its cellsare modified at the blastocyst state, or genomic modification can takeplace when a single-cell is modified. If a particular gene isinactivated by a knock out modification, homozygosity would normally berequired. If a particular gene is inactivated by an RNA interference ordominant negative strategy, then heterozygosity is often adequate.

Typically, in embryo/zygote microinjection, a nucleic acid construct ormRNA is introduced into a fertilized egg; 1 or 2 cell fertilized eggsare used as the pronuclei containing the genetic material from the spermhead and the egg are visible within the protoplasm. Pronuclear stagedfertilized eggs can be obtained in vitro or in vivo (i.e., surgicallyrecovered from the oviduct of donor animals). In vitro fertilized eggscan be produced as follows. For example, swine ovaries can be collectedat an abattoir, and maintained at 22-28° C. during transport. Ovariescan be washed and isolated for follicular aspiration, and folliclesranging from 4-8 mm can be aspirated into 50 mL conical centrifuge tubesusing 18 gauge needles and under vacuum. Follicular fluid and aspiratedoocytes can be rinsed through pre-filters with commercial TL-HEPES(Minitube, Verona, Wis.). Oocytes surrounded by a compact cumulus masscan be selected and placed into TCM-199 OOCYTE MATURATION MEDIUM(Minitube) supplemented with 0.1 mg/mL cysteine, 10 ng/mL epidermalgrowth factor, 10% porcine follicular fluid, 50 μM 2-mercaptoethanol,0.5 mg/ml cAMP, 10 IU/mL each of pregnant mare serum gonadotropin (PMSG)and human chorionic gonadotropin (hCG) for approximately 22 hours inhumidified air at 38.7° C. and 5% CO₂. Subsequently, the oocytes can bemoved to fresh TCM-199 maturation medium, which will not contain cAMP,PMSG or hCG and incubated for an additional 22 hours. Matured oocytescan be stripped of their cumulus cells by vortexing in 0.1%hyaluronidase for 1 minute.

For swine, mature oocytes can be fertilized in 500 μl Minitube PORCPROIVF MEDIUM SYSTEM (Minitube) in Minitube 5-well fertilization dishes. Inpreparation for in vitro fertilization (IVF), freshly-collected orfrozen boar semen can be washed and resuspended in PORCPRO IVF Medium to433 10×5 sperm. Sperm concentrations can be analyzed by computerassisted semen analysis (SPERMVISION, Minitube). Final in vitroinsemination can be performed in a 10 μl volume at a final concentrationof approximately 40 motile sperm/oocyte, depending on boar. Incubate allfertilizing oocytes at 38.7° C. in 5.0% CO₂ atmosphere for 6 hours. Sixhours post-insemination, presumptive zygotes can be washed twice inNCSU-23 and moved to 0.5 mL of the same medium. This system can produce20-30% blastocysts routinely across most boars with a 10-30% polyspermicinsemination rate.

Linearized nucleic acid constructs or mRNA can be injected into one ofthe pronuclei or into the cytoplasm. Then the injected eggs can betransferred to a recipient female (e.g., into the oviducts of arecipient female) and allowed to develop in the recipient female toproduce the transgenic animals. In particular, in vitro fertilizedembryos can be centrifuged at 15,000×g for 5 minutes to sediment lipidsallowing visualization of the pronucleus. The embryos can be injectedwith using an Eppendorf FEMTOJET injector and can be cultured untilblastocyst formation. Rates of embryo cleavage and blastocyst formationand quality can be recorded.

Embryos can be surgically transferred into uteri of asynchronousrecipients. Typically, 100-200 (e.g., 150-200) embryos can be depositedinto the ampulla-isthmus junction of the oviduct using a 5.5-inchTOMCAT® catheter. After surgery, real-time ultrasound examination ofpregnancy can be performed.

In somatic cell nuclear transfer, a transgenic cell (e.g., a transgenicpig cell or bovine cell) such as an embryonic blastomere, fetalfibroblast, adult ear fibroblast, or granulosa cell that includes anucleic acid construct described above, can be introduced into anenucleated oocyte to establish a combined cell. Oocytes can beenucleated by partial zona dissection near the polar body and thenpressing out cytoplasm at the dissection area. Typically, an injectionpipette with a sharp beveled tip is used to inject the transgenic cellinto an enucleated oocyte arrested at meiosis 2. In some conventions,oocytes arrested at meiosis-2 are termed eggs. After producing a porcineor bovine embryo (e.g., by fusing and activating the oocyte), the embryois transferred to the oviducts of a recipient female, about 20 to 24hours after activation. See, for example, Cibelli et al. (1998) Science280, 1256-1258 and U.S. Pat. No. 6,548,741. For pigs, recipient femalescan be checked for pregnancy approximately 20-21 days after transfer ofthe embryos.

Standard breeding techniques can be used to create animals that arehomozygous for the edited nucleic acid from the initial heterozygousfounder animals. Homozygosity may not be required, however. Transgenicpigs described herein can be bred with other pigs of interest.

Once transgenic animals have been generated, presence of the editednucleic acid can be assessed using standard techniques. Initialscreening can be accomplished by sequencing or Southern blot analysis.For a description of Southern analysis, see sections 9.37-9.52 ofSambrook et al., 1989, Molecular Cloning, A Laboratory Manual, secondedition, Cold Spring Harbor Press, Plainview, N.Y. Polymerase chainreaction (PCR) techniques also can be used in the initial screening. PCRrefers to a procedure or technique in which target nucleic acids areamplified. Generally, sequence information from the ends of the regionof interest or beyond are employed to design oligonucleotide primersthat are identical or similar in sequence to opposite strands of thetemplate to be amplified. PCR can be used to amplify specific sequencesfrom DNA as well as RNA, including sequences from total genomic DNA ortotal cellular RNA. Primers typically are 14 to 40 nucleotides inlength, but can range from 10 nucleotides to hundreds of nucleotides inlength. PCR is described in, for example PCR Primer: A LaboratoryManual, ed. Dieffenbach and Dveksler, Cold Spring Harbor LaboratoryPress, 1995. Nucleic acids also can be amplified by ligase chainreaction, strand displacement amplification, self-sustained sequencereplication, or nucleic acid sequence-based amplified. See, for example,Lewis (1992) Genetic Engineering News 12,1; Guatelli et al. (1990) Proc.Natl. Acad. Sci. USA 87:1874; and Weiss (1991) Science 254:1292. At theblastocyst stage, embryos can be individually processed for analysis byPCR, Southern hybridization and splinkerette PCR (see, e.g., Dupuy etal. Proc Natl Acad Sci USA (2002) 99:4495).

Expression of a nucleic acid sequence encoding a polypeptide in thetissues of transgenic pigs can be assessed using techniques thatinclude, for example, Northern blot analysis of tissue samples obtainedfrom the animal, in situ hybridization analysis, Western analysis,immunoassays such as enzyme-linked immunosorbent assays, andreverse-transcriptase PCR (RT-PCR).

Interfering RNAs

A variety of interfering RNA (RNAi) systems are known. Double-strandedRNA (dsRNA) induces sequence-specific degradation of homologous genetranscripts. RNA-induced silencing complex (RISC) metabolizes dsRNA tosmall 21-23-nucleotide small interfering RNAs (siRNAs). RISC contains adouble stranded RNAse (dsRNase, e.g., Dicer) and ssRNase (e.g., Argonaut2 or Ago2). RISC utilizes antisense strand as a guide to find acleavable target. Both siRNAs and microRNAs (miRNAs) are known. A methodof inactivating a gene in a genetically edited animal comprises inducingRNA interference against a target gene and/or nucleic acid such thatexpression of the target gene and/or nucleic acid is reduced.

For example, the exogenous nucleic acid sequence can induce RNAinterference against a nucleic acid encoding a polypeptide. For example,double-stranded small interfering RNA (siRNA) or small hairpin RNA(shRNA) homologous to a target DNA can be used to reduce expression ofthat DNA. Constructs for siRNA can be produced as described, forexample, in Fire et al. (1998) Nature 391:806; Romano and Masino (1992)Mol. Microbiol. 6:3343; Cogoni et al. (1996) EMBO J. 15:3153; Cogoni andMasino (1999) Nature 399:166; Misquitta and Paterson (1999) Proc. Natl.Acad. Sci. USA 96:1451; and Kennerdell and Carthew (1998) Cell 95:1017.Constructs for shRNA can be produced as described by McIntyre andFanning (2006) BMC Biotechnology 6:1. In general, shRNAs are transcribedas a single-stranded RNA molecule containing complementary regions,which can anneal and form short hairpins.

The probability of finding a single, individual functional siRNA ormiRNA directed to a specific gene is high. The predictability of aspecific sequence of siRNA, for instance, is about 50% but a number ofinterfering RNAs may be made with good confidence that at least one ofthem will be effective.

Embodiments include an in vitro cell, an in vivo cell, and a geneticallyedited animal such as a livestock animal that express an RNAi directedagainst a somatostatin receptor gene selective for improved growthperformance. An embodiment is an RNAi directed against a gene selectedfrom SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5. The RNAi may be, forinstance, selected from the group consisting of siRNA, shRNA, dsRNA,RISC and miRNA.

Inducible Systems

An inducible system may be used to control expression of a somatostatinreceptor gene. Various inducible systems are known that allowspatiotemporal control of expression of a gene. Several have been provento be functional in vivo in transgenic animals.

An example of an inducible system is the tetracycline (tet)-on promotersystem, which can be used to regulate transcription of the nucleic acid.In this system, a mutated Tet repressor (TetR) is fused to theactivation domain of herpes simplex virus VP 16 trans-activator proteinto create a tetracycline-controlled transcriptional activator (tTA),which is regulated by tet or doxycycline (dox). In the absence ofantibiotic, transcription is minimal, while in the presence of tet ordox, transcription is induced. Alternative inducible systems include theecdysone or rapamycin systems. Ecdysone is an insect molting hormonewhose production is controlled by a heterodimer of the ecdysone receptorand the product of the ultraspiracle gene (USP). Expression is inducedby treatment with ecdysone or an analog of ecdysone such as muristeroneA. The agent that is administered to the animal to trigger the induciblesystem is referred to as an induction agent.

The tetracycline-inducible system and the Cre/loxP recombinase system(either constitutive or inducible) are among the more commonly usedinducible systems. The tetracycline-inducible system involves atetracycline-controlled transactivator (tTA)/reverse tTA (rtTA). Amethod to use these systems in vivo involves generating two lines ofgenetically edited animals. One animal line expresses the activator(tTA, rtTA, or Cre recombinase) under the control of a selectedpromoter. Another set of transgenic animals express the acceptor, inwhich the expression of the gene of interest (or the gene to bemodified) is under the control of the target sequence for the tTA/rtTAtransactivators (or is flanked by loxP sequences). Mating the twostrains of mice provides control of gene expression.

The tetracycline-dependent regulatory systems (tet systems) rely on twocomponents, i.e., a tetracycline-controlled transactivator (tTA or rtTA)and a tTA/rtTA-dependent promoter that controls expression of adownstream cDNA, in a tetracycline-dependent manner. In the absence oftetracycline or its derivatives (such as doxycycline), tTA binds to tetOsequences, allowing transcriptional activation of the tTA-dependentpromoter. However, in the presence of doxycycline, tTA cannot interactwith its target and transcription does not occur. The tet system thatuses tTA is termed tet-OFF, because tetracycline or doxycycline allowstranscriptional down-regulation. Administration of tetracycline or itsderivatives allows temporal control of transgene expression in vivo.rtTA is a variant of tTA that is not functional in the absence ofdoxycycline but requires the presence of the ligand for transactivation.This tet system is therefore termed tet-ON. The tet systems have beenused in vivo for the inducible expression of several transgenes,encoding, e.g., reporter genes, oncogenes, or proteins involved in asignaling cascade.

The Cre/lox system uses the Cre recombinase, which catalyzessite-specific recombination by crossover between two distant Crerecognition sequences, i.e., loxP sites. A DNA sequence introducedbetween the two loxP sequences (termed foxed DNA) is excised byCre-mediated recombination. Control of Cre expression in a transgenicanimal, using either spatial control (with a tissue- or cell-specificpromoter), or temporal control (with an inducible system), results incontrol of DNA excision between the two loxP sites. One application isfor conditional gene inactivation (conditional knockout). Anotherapproach is for protein over-expression, wherein a foxed stop codon isinserted between the promoter sequence and the DNA of interest.Genetically edited animals do not express the transgene until Cre isexpressed, leading to excision of the floxed stop codon. This system hasbeen applied to tissue-specific oncogenesis and controlled antigenereceptor expression in B lymphocytes. Inducible Cre recombinases havealso been developed. The inducible Cre recombinase is activated only byadministration of an exogenous ligand. The inducible Cre recombinasesare fusion proteins containing the original Cre recombinase and aspecific ligand-binding domain. The functional activity of the Crerecombinase is dependent on an external ligand that is able to bind tothis specific domain in the fusion protein.

Embodiments include an in vitro cell, an in vivo cell, and a geneticallyedited animal such as a livestock animal that comprise a somatostatinreceptor gene selective for improved growth performance that is undercontrol of an inducible system. The genetic modification of an animalmay be genomic or mosaic. An embodiment is a gene in the groupconsisting of SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 that is undercontrol of an inducible system. The inducible system may be, forinstance, selected from the group consisting of Tet-On, Tet-Off,Cre-lox, and Hif1 alpha.

Vectors and Nucleic Acids

A variety of nucleic acids may be introduced into cells for knockoutpurposes, for inactivation of a gene, to obtain expression of a gene, orfor other purposes. As used herein, the term nucleic acid includes DNA,RNA, and nucleic acid analogs, and nucleic acids that aredouble-stranded or single-stranded (i.e., a sense or an antisense singlestrand). Nucleic acid analogs can be modified at the base moiety, sugarmoiety, or phosphate backbone to improve, for example, stability,hybridization, or solubility of the nucleic acid. Modifications at thebase moiety include deoxyuridine for deoxythymidine, and5-methyl-2′-deoxycytidine and 5-bromo-2′-doxycytidine for deoxycytidine.Modifications of the sugar moiety include modification of the 2′hydroxyl of the ribose sugar to form 2′-O-methyl or 2′-O-allyl sugars.The deoxyribose phosphate backbone can be modified to produce morpholinonucleic acids, in which each base moiety is linked to a six membered,morpholino ring, or peptide nucleic acids, in which the deoxyphosphatebackbone is replaced by a pseudopeptide backbone and the four bases areretained. See, Summerton and Weller (1997) Antisense Nucleic Acid DrugDev. 7(3):187; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5. Inaddition, the deoxyphosphate backbone can be replaced with, for example,a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite,or an alkyl phosphotriester backbone.

The target nucleic acid sequence can be operably linked to a regulatoryregion such as a promoter. Regulatory regions can be porcine regulatoryregions or can be from other species. As used herein, operably linkedrefers to positioning of a regulatory region relative to a nucleic acidsequence in such a way as to permit or facilitate transcription of thetarget nucleic acid.

Any type of promoter can be operably linked to a target nucleic acidsequence. Examples of promoters include, without limitation,tissue-specific promoters, constitutive promoters, inducible promoters,and promoters responsive or unresponsive to a particular stimulus.Suitable tissue specific promoters can result in preferential expressionof a nucleic acid transcript in beta cells and include, for example, thehuman insulin promoter. Other tissue specific promoters can result inpreferential expression in, for example, hepatocytes or heart tissue andcan include the albumin or alpha-myosin heavy chain promoters,respectively. In other embodiments, a promoter that facilitates theexpression of a nucleic acid molecule without significant tissue ortemporal-specificity can be used (i.e., a constitutive promoter). Forexample, a beta-actin promoter such as the chicken beta-actin genepromoter, ubiquitin promoter, miniCAGs promoter,glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, or3-phosphoglycerate kinase (PGK) promoter can be used, as well as viralpromoters such as the herpes simplex virus thymidine kinase (HSV-TK)promoter, the SV40 promoter, or a cytomegalovirus (CMV) promoter. Insome embodiments, a fusion of the chicken beta actin gene promoter andthe CMV enhancer is used as a promoter. See, for example, Xu et al.(2001) Hum. Gene Ther. 12:563; and Kiwaki et al. (1996) Hum. Gene Ther.7:821.

Additional regulatory regions that may be useful in nucleic acidconstructs, include, but are not limited to, polyadenylation sequences,translation control sequences (e.g., an internal ribosome entry segment,IRES), enhancers, inducible elements, or introns. Such regulatoryregions may not be necessary, although they may increase expression byaffecting transcription, stability of the mRNA, translationalefficiency, or the like. Such regulatory regions can be included in anucleic acid construct as desired to obtain optimal expression of thenucleic acids in the cell(s). Sufficient expression, however, cansometimes be obtained without such additional elements.

A nucleic acid construct may be used that encodes signal peptides orselectable markers. Signal peptides can be used such that an encodedpolypeptide is directed to a particular cellular location (e.g., thecell surface). Non-limiting examples of selectable markers includepuromycin, ganciclovir, adenosine deaminase (ADA), aminoglycosidephosphotransferase (neo, G418, APH), dihydrofolate reductase (DHFR),hygromycin-B-phosphtransferase, thymidine kinase (TK), andxanthin-guanine phosphoribosyltransferase (XGPRT). Such markers areuseful for selecting stable transformants in culture. Other selectablemarkers include fluorescent polypeptides, such as green fluorescentprotein or yellow fluorescent protein.

In some embodiments, a sequence encoding a selectable marker can beflanked by recognition sequences for a recombinase such as, e.g., Cre orFlp. For example, the selectable marker can be flanked by loxPrecognition sites (34-bp recognition sites recognized by the Crerecombinase) or FRT recognition sites such that the selectable markercan be excised from the construct. See, Orban, et al., Proc. Natl. Acad.Sci. (1992) 89:6861, for a review of Cre/lox technology, and Brand andDymecki, Dev. Cell (2004) 6:7. A transposon containing a Cre- orFlp-activatable transgene interrupted by a selectable marker gene alsocan be used to obtain transgenic animals with conditional expression ofa transgene. For example, a promoter driving expression of themarker/transgene can be either ubiquitous or tissue-specific, whichwould result in the ubiquitous or tissue-specific expression of themarker in F0 animals (e.g., pigs). Tissue specific activation of thetransgene can be accomplished, for example, by crossing a pig thatubiquitously expresses a marker-interrupted transgene to a pigexpressing Cre or Flp in a tissue-specific manner, or by crossing a pigthat expresses a marker-interrupted transgene in a tissue-specificmanner to a pig that ubiquitously expresses Cre or Flp recombinase.Controlled expression of the transgene or controlled excision of themarker allows expression of the transgene.

In some embodiments, the exogenous nucleic acid encodes a polypeptide. Anucleic acid sequence encoding a polypeptide can include a tag sequencethat encodes a “tag” designed to facilitate subsequent manipulation ofthe encoded polypeptide (e.g., to facilitate localization or detection).Tag sequences can be inserted in the nucleic acid sequence encoding thepolypeptide such that the encoded tag is located at either the carboxylor amino terminus of the polypeptide. Non-limiting examples of encodedtags include glutathione S-transferase (GST) and FLAG™ag (Kodak, NewHaven, Conn.).

Nucleic acid constructs can be methylated using an SssI CpG methylase(New England Biolabs, Ipswich, Mass.). In general, the nucleic acidconstruct can be incubated with S-adenosylmethionine and SssICpG-methylase in buffer at 37° C. Hypermethylation can be confirmed byincubating the construct with one unit of HinP1I endonuclease for 1 hourat 37° C. and assaying by agarose gel electrophoresis.

Nucleic acid constructs can be introduced into embryonic, fetal, oradult animal cells of any type, including, for example, germ cells suchas an oocyte or an egg, a progenitor cell, an adult or embryonic stemcell, a primordial germ cell, a kidney cell such as a PK-15 cell, anislet cell, a beta cell, a liver cell, or a fibroblast such as a dermalfibroblast, using a variety of techniques. Non-limiting examples oftechniques include the use of transposon systems, recombinant virusesthat can infect cells, or liposomes or other non-viral methods such aselectroporation, microinjection, or calcium phosphate precipitation,that are capable of delivering nucleic acids to cells.

In transposon systems, the transcriptional unit of a nucleic acidconstruct, i.e., the regulatory region operably linked to an exogenousnucleic acid sequence, is flanked by an inverted repeat of a transposon.Several transposon systems, including, for example, Sleeping Beauty(see, U.S. Pat. No. 6,613,752 and U.S. Publication No. 2005/0003542);Frog Prince (Miskey et al. (2003) Nucleic Acids Res. 31:6873); Tol2(Kawakami (2007) Genome Biology 8 (Suppl.1):S7; Minos (Pavlopoulos etal. (2007) Genome Biology 8 (Suppl.1):S2); Hsmar1 (Miskey et al. (2007))Mol Cell Biol. 27:4589); and Passport have been developed to introducenucleic acids into cells, including mice, human, and pig cells. TheSleeping Beauty transposon is particularly useful. A transposase can bedelivered as a protein, encoded on the same nucleic acid construct asthe exogenous nucleic acid, can be introduced on a separate nucleic acidconstruct, or provided as an mRNA (e.g., an in vitro-transcribed andcapped mRNA).

Insulator elements also can be included in a nucleic acid construct tomaintain expression of the exogenous nucleic acid and to inhibit theunwanted transcription of host genes. See, for example, U.S. PublicationNo. 2004/0203158. Typically, an insulator element flanks each side ofthe transcriptional unit and is internal to the inverted repeat of thetransposon. Non-limiting examples of insulator elements include thematrix attachment region-(MAR) type insulator elements and border-typeinsulator elements. See, for example, U.S. Pat. Nos. 6,395,549,5,731,178, 6,100,448, and 5,610,053, and U.S. Publication No.2004/0203158.

Nucleic acids can be incorporated into vectors. A vector is a broad termthat includes any specific DNA segment that is designed to move from acarrier into a target DNA. A vector may be referred to as an expressionvector, or a vector system, which is a set of components needed to bringabout DNA insertion into a genome or other targeted DNA sequence such asan episome, plasmid, or even virus/phage DNA segment. Vector systemssuch as viral vectors (e.g., retroviruses, adeno-associated virus andintegrating phage viruses), and non-viral vectors (e.g., transposons)used for gene delivery in animals have two basic components: 1) a vectorcomprised of DNA (or RNA that is reverse transcribed into a cDNA) and 2)a transposase, recombinase, or other integrase enzyme that recognizesboth the vector and a DNA target sequence and inserts the vector intothe target DNA sequence. Vectors most often contain one or moreexpression cassettes that comprise one or more expression controlsequences, wherein an expression control sequence is a DNA sequence thatcontrols and regulates the transcription and/or translation of anotherDNA sequence or mRNA, respectively.

Many different types of vectors are known. For example, plasmids andviral vectors, e.g., retroviral vectors, are known. Mammalian expressionplasmids typically have an origin of replication, a suitable promoterand optional enhancer, necessary ribosome binding sites, apolyadenylation site, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking non-transcribed sequences.Examples of vectors include: plasmids (which may also be a carrier ofanother type of vector), adenovirus, adeno-associated virus (AAV),lentivirus (e.g., modified HIV-1, SIV or FIV), retrovirus (e.g., ASV,ALV or MoMLV), and transposons (e.g., Sleeping Beauty, P-elements,Tol-2, Frog Prince, piggyBac).

Founder Animals, Animal Lines, Traits, and Reproduction

Founder animals may be produced by cloning and other methods describedherein. The founders can be homozygous for a genetic modification, as inthe case where a zygote or a primary cell undergoes a homozygousmodification. Similarly, founders can also be made that areheterozygous. The founders may be genomically modified, meaning that allof the cells in their genome have undergone modification. Founders canbe mosaic for a modification, as may happen when vectors are introducedinto one of a plurality of cells in an embryo, typically at a blastocyststage. Progeny of mosaic animals may be tested to identify progeny thatare genomically modified. An animal line is established when a pool ofanimals has been created that can be reproduced sexually or by assistedreproductive techniques, with heterogeneous or homozygous progenyconsistently expressing the modification.

A further embodiment includes a method for screening animals todetermine those more likely to exhibit improved growth performance.These methods include obtaining a genetic sample from the animal. Themethods can further include assaying for the presence or absence of amodified somatostatin receptor gene associated with improved growthperformance.

Further embodiments of the invention can include amplifying the gene ora region of the gene, which contains at least one modification. Sinceone of the modifications may involve changes in the amino acidcomposition of the somatostatin receptor protein, assay methods may eveninvolve ascertaining the amino acid composition of these proteins.Methods for this type or purification and analysis typically involveisolation of the protein through means including fluorescence taggingwith antibodies, separation and purification of the protein (i.e.,through reverse phase HPLC system), and use of an automated proteinsequencer to identify the amino acid sequence present. Protocols forthis assay are standard and known in the art and are disclosed inAusubel et al. (eds.), Short Protocols in Molecular Biology 4^(th) ed.(John Wiley and Sons 1999).

A further embodiment comprises a breeding method whereby assays of theabove types are conducted on a plurality of gene sequences fromdifferent animals or animal embryos of various species to be selectedfrom and, based on the results, certain animals are either selected ordropped out of the breeding program.

All references, including publications, patents, and patentapplications, cited herein are hereby incorporated by reference to theextent they are not inconsistent with the explicit details of thisdisclosure, and are so incorporated to the same extent as if eachreference were individually and specifically indicated to beincorporated by reference and were set forth in its entirety herein. Thereferences discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention. The followingexamples are provided to illustrate certain particular features and/orembodiments. The examples should not be construed to limit thedisclosure to the particular features or embodiments exemplified.

EXAMPLES Example 1

A surgical embryo transfer, containing somatic cell nuclear transfer andIVF derived embryos that were injected with CRISPR/Cas9 molecules todisrupt the SSTR2 locus (Cas9 mRNA and gRNA that targeted a locus inSSTR2 (SEQ ID NO: 5)), was performed. Two male piglets (74-1 and 74-2)were born alive. Sequencing data (FIGS. 1-3) shows that both pigs areclones resulting from somatic cell nuclear transfer and carry a compoundheterozygous mutation with 1 bp or 3 bp deletions in the SSTR2 gene (SEQID NOs: 3 and 4). Body weights for 74-1 are above average and 74-2 arewell above average for normal piglet growth during lactation (FIG. 4).

>Wild type Sus scrofa SSTR2 nucleotide sequence (SEQ ID NO: 1)atggatatggcgtatgagctactcaacgggagccagccgtggctttcctctccattcgacctcaatggctccgtggcaacagccaacagttcaaaccagacggagccatactatgacctgaccagcaatgcagtcctcacgttcatatattttgtggtctgcatcattggcctgtgcggcaacacgcttgtcatttacgtcatcctccgctacgccaagatgaagacaatcaccaacatctacatcctcaacctggccattgccgatgagctcttcatgctgggcctgcccttcctggccatgcaggtggctctggtccactggccctttggcaaggccatctgccgggtggtcatgactgtggatggcatcaatcagttcaccagcattttctgcttgaccgtcatgagcattgaccggtacctggctgtggtccaccccatcaagtcggccaagtggaggagaccccggacagccaagatgatcaatgtggccgtgtggggcgtctctctgctggtcatcttgcccatcatgatatatgccgggcttcgaagcaaccagtgggggagaagcagctgcaccatcaactggccaggcgagtcgggggcatggtacacggggttcattatctacgccttcatcctggggttcctggtgcccctcaccatcatctgtctttgctacctgttcattatcatcaaggtgaagtcctccggaatccgagtgggttcctccaagaggaaaaagtctgagaagaaggtcacccggatggtgtccattgtggtggccgtcttcattttctgctggctccccttctacatcttcaatgtctcttcggtctctgtggccatcagtcccaccccagcccttaaaggcatgtttgactttgtggtggtcctcacctatgctaacagctgtgccaaccctatcctctatgccttcttgtccgacaacttcaagaagagcttccagaatgtcctctgcttggtcaaggtgagcggcacagatgatggggaacggagtgacagtaagcaggacaaatcgcggctgaatgagaccacggagacccagaggaccctcctcaatggagacctccagac cagtatctga>Wild type Sus scrofa SSTR2 protein sequence (SEQ ID NO: 2)MDMAYELLNGSQPWLSSPFDLNGSVATANSSNQTEPYYDLTSNAVLTFIYFVVCIIGLCGNTLVIYVILRYAKMKTITNIYILNLAIADELFMLGLPFLAMQVALVHWPFGKAICRVVMTVDGINQFTSIFCLTVMSIDRYLAVVHPIKSAKWRRPRTAKMINVAVWGVSLLVILPIMIYAGLRSNQWGRSSCTINWPGESGAWYTGFIIYAFILGFLVPLTIICLCYLFIIIKVKSSGIRVGSSKRKKSEKKVTRMVSIVVAVFIFCWLPFYIFNVSSVSVAISPTPALKGMFDFVVVLTYANSCANPILYAFLSDNFKKSFQNVLCLVKVSGTDDGERSDSKQDKSRL NETTETQRTLLNGDLQTSI>Modified Sus scrofa SSTR2 nucleotide sequence (SEQ ID NO: 3)atggatatggcgtatgagctaaacgggagccagccgtggctttcctctccattcgacctcaatggctccgtggcaacagccaacagttcaaaccagacggagccatactatgacctgaccagcaatgcagtcctcacgttcatatattttgtggtctgcatcattggcctgtgcggcaacacgcttgtcatttacgtcatcctccgctacgccaagatgaagacaatcaccaacatctacatcctcaacctggccattgccgatgagctcttcatgctgggcctgcccttcctggccatgcaggtggctctggtccactggccctttggcaaggccatctgccgggtggtcatgactgtggatggcatcaatcagttcaccagcattttctgcttgaccgtcatgagcattgaccggtacctggctgtggtccaccccatcaagtcggccaagtggaggagaccccggacagccaagatgatcaatgtggccgtgtggggcgtctctctgctggtcatcttgcccatcatgatatatgccgggcttcgaagcaaccagtgggggagaagcagctgcaccatcaactggccaggcgagtcgggggcatggtacacggggttcattatctacgccttcatcctggggttcctggtgcccctcaccatcatctgtctttgctacctgttcattatcatcaaggtgaagtcctccggaatccgagtgggttcctccaagaggaaaaagtctgagaagaaggtcacccggatggtgtccattgtggtggccgtcttcattttctgctggctccccttctacatcttcaatgtctcttcggtctctgtggccatcagtcccaccccagcccttaaaggcatgtttgactttgtggtggtcctcacctatgctaacagctgtgccaaccctatcctctatgccttcttgtccgacaacttcaagaagagcttccagaatgtcctctgcttggtcaaggtgagcggcacagatgatggggaacggagtgacagtaagcaggacaaatcgcggctgaatgagaccacggagacccagaggaccctcctcaatggagacctccagaccag tatctga>Modified Sus scrofa SSTR2 nucleotide sequence (SEQ ID NO: 4)atggatatggcgtatgagctactcacgggagccagccgtggctttcctctccattcgacctcaatggctccgtggcaacagccaacagttcaaaccagacggagccatactatgacctgaccagcaatgcagtcctcacgttcatatattttgtggtctgcatcattggcctgtgcggcaacacgcttgtcatttacgtcatcctccgctacgccaagatgaagacaatcaccaacatctacatcctcaacctggccattgccgatgagctcttcatgctgggcctgcccttcctggccatgcaggtggctctggtccactggccctttggcaaggccatctgccgggtggtcatgactgtggatggcatcaatcagttcaccagcattttctgcttgaccgtcatgagcattgaccggtacctggctgtggtccaccccatcaagtcggccaagtggaggagaccccggacagccaagatgatcaatgtggccgtgtggggcgtctctctgctggtcatcttgcccatcatgatatatgccgggcttcgaagcaaccagtgggggagaagcagctgcaccatcaactggccaggcgagtcgggggcatggtacacggggttcattatctacgccttcatcctggggttcctggtgcccctcaccatcatctgtctttgctacctgttcattatcatcaaggtgaagtcctccggaatccgagtgggttcctccaagaggaaaaagtctgagaagaaggtcacccggatggtgtccattgtggtggccgtcttcattttctgctggctccccttctacatcttcaatgtctcttcggtctctgtggccatcagtcccaccccagcccttaaaggcatgtttgactttgtggtggtcctcacctatgctaacagctgtgccaaccctatcctctatgccttcttgtccgacaacttcaagaagagcttccagaatgtcctctgcttggtcaaggtgagcggcacagatgatggggaacggagtgacagtaagcaggacaaatcgcggctgaatgagaccacggagacccagaggaccctcctcaatggagacctccagaccagtatctga >SSTR2 Guide RNA (SEQ ID NO: 5) tggcgtatgagctactcaac

Example 2

F₁ litters were generated by crossing the founder boar, 74-02, withthree wild-type gilts. All of the piglets were heterozygous, carryingone wild type copy of the SSTR2 allele and one copy of either the 1 bpdeletion (n=22) or 3 bp deletion (n=24) allele. All of the genotypeswere confirmed with Sanger sequencing, and a subset were furtherverified by sequencing clones produced using TA cloning. Pigs wereweaned at 21 days of age (1 bp deletion, n=16; 3 bp deletion, n=20) andwere group housed with litter mates. After weaning, all pigs had adlibitum access to feed and water. Weights were recorded for eachindividual pig at birth and once every week through 49 days of age.Weights at each time point were analyzed using a mixed model in SAS 9.4with dam as a random effect.

FIG. 6 shows weekly weight data comparing heterozygous males carryingthe 1 bp deletion and heterozygous males carrying the 3 bp deletion. Nodifferences were observed between groups at birth, but differences wereobserved at all other time points. FIG. 7 shows weekly weight datacomparing heterozygous females carrying the 1 bp deletion andheterozygous females carrying the 3 bp deletion. No differences wereobserved between groups at any of the time points.

What is claimed is:
 1. A genetically edited livestock animal or progenythereof comprising an edited chromosomal sequence that alters expressionor activity of a somatostatin receptor (SSTR) protein.
 2. Thegenetically edited livestock animal of claim 1, wherein the editedchromosomal sequence comprises a substitution, insertion, or deletion ofone or more nucleotides in the SSTR coding sequence.
 3. The geneticallyedited livestock animal of claim 1, wherein the edited chromosomalsequence reduces or eliminates the expression or activity of the SSTRprotein.
 4. The genetically edited livestock animal of claim 1, whereinthe edited chromosomal sequence comprises no exogenously introducedsequence.
 5. The genetically edited livestock animal of claim 1, whereinthe SSTR protein is SSTR1, SSTR2, SSTR3, SSTR4, or SSTR5.
 6. Thegenetically edited livestock animal of claim 1, wherein the SSTR proteinis SSTR2.
 7. The genetically edited livestock animal of claim 1, whereinthe animal is heterozygous or homozygous for the edited chromosomalsequence.
 8. The genetically edited livestock animal of claim 1, whereinthe animal is a pig.
 9. The genetically edited livestock animal of claim1, wherein the edited chromosomal sequence comprises SEQ ID NO: 3 or 4.10. The genetically edited livestock animal of claim 1, wherein theedited chromosomal sequence improves growth performance of the livestockanimal.
 11. A genetically edited livestock animal cell comprising anedited chromosomal sequence that alters expression or activity of asomatostatin receptor (SSTR) protein.
 12. The genetically editedlivestock animal cell of claim 11, wherein the edited chromosomalsequence comprises a substitution, insertion, or deletion of one or morenucleotides in the SSTR coding sequence.
 13. The genetically editedlivestock animal cell of claim 11, wherein the edited chromosomalsequence reduces or eliminates the expression or activity of the SSTRprotein.
 14. The genetically edited livestock animal cell of claim 11,wherein the SSTR protein is SSTR1, SSTR2, SSTR3, SSTR4, or SSTR5. 15.The genetically edited livestock animal cell of claim 11, wherein theSSTR protein is SSTR2.
 16. The genetically edited livestock animal cellof claim 11, wherein the cell is heterozygous or homozygous for theedited chromosomal sequence.
 17. The genetically edited livestock animalcell of claim 11, further comprising a conditional knock-out system forconditional expression of the SSTR protein.
 18. The genetically editedlivestock animal cell of claim 11, wherein the cell is from a pig. 19.The genetically edited livestock animal cell of claim 11, wherein thecell is a sperm cell or an egg cell.
 20. The genetically editedlivestock animal cell of claim 11, wherein the cell is a somatic cell.21. The genetically edited livestock animal cell of claim 11, whereinthe edited chromosomal sequence comprises SEQ ID NO: 3 or
 4. 22. Amethod of generating a livestock animal with improved growth performancecomprising: editing a chromosomal sequence of the animal to create amodification which reduces or eliminates the expression or activity of asomatostatin receptor (SSTR) protein.
 23. The method of claim 22,wherein the editing is by use of a TALEN, a zinc finger nuclease, or aCRISPR system.
 24. The method of claim 22, wherein the generatingcomprises use of somatic cell nuclear transfer.
 25. The method of claim22, wherein the edited chromosomal sequence comprises a substitution,insertion, or deletion of one or more nucleotides in the SSTR codingsequence.
 26. The method of claim 22, wherein the SSTR protein is SSTR1,SSTR2, SSTR3, SSTR4, or SSTR5.
 27. The method of claim 22, wherein theSSTR protein is SSTR2.
 28. The method of claim 22, wherein the animal isa pig.